Heretofore, thermophilic microbes have been made to act on organic wastes such as droppings of domestic animals, excrement and urine, sludge, and city waste to aerobically ferment the wastes and to make them odorless and dry, thereby preparing compost. Further, as such thermophilic microbes, there have been known thermophilic actinomycetes belonging to the genera Thermoactinomyces or Thermomonospora (JP 55-121992 A), a mixture of thermophilic, aerobic and spore-forming bacteria such as those belonging to the genera Bacillus or Geobacillus or lactic acid-producing bacteria (JP 51-129759 A), aerobic Bacillus subtilis (JP 6-5197 A), bacteria belonging to the genus Thermus aquaticus having lignin-solubilizing capability (JP 6-105679 A), aerobic cellulose degrading bacteria Clostridium thermocellum, Thermus aquaticus (JP 6-191977) and so forth.
However, in spite of use of these microbes, although the fermentation temperature is elevated to 70° C. or more due to fermentation heat at the time of fermentation, the temperature is elevated up to 80° C. at the most, and thus, saprophytes, in particular, spore-forming saprophytes could not be extinguished. In addition, the number of useful bacteria cells in the obtained fertilizer is at most around 100,000,000 per g (dry fertilizer), so that when the cells are used as a fertilizer, the fertilizing effect could not be exhibited sufficiently.
To solve these problems regarding the disposal of sludge, the inventors of the present invention have made intensive studies to obtain a fermented product which purifies the sludge by subjecting the sludge to fermentation treatment at high temperatures of 85° C. or more, more preferably 95° C. or more, to extinguish saprophytes, seed of weeds, and the like, and which contains a large number of useful bacteria cells. As a result, the inventors found a method of obtaining a fermented sludge product containing a large number of solely the useful bacteria cells, comprising: adding a culture of a bacterium that grows at temperatures not lower than 85° C. obtained from the soil of Kirishima volcanic region in the prefecture of Kagoshima, Japan to raw sludge and mixing them; and subjecting the resulting mixture to aerobic fermentation to extinguish the saprophytes and seeds contained in the sludge at a fermentation temperature of 85° C. or more to purify the sludge, and have obtained a patent on the method (JP 3064221 B). And, the fermented sludge has been used as compost, in which mesophilic aerobic spore-forming bacteria, thermophilic aerobic, spore-forming bacteria, thermophiles and the like belonging to the genera Bacillus or Geobacillus have been found in large numbers.
That is, about 1,000,000,000 bacteria cells are included per g of the fermented sludge, the bacteria predominantly including aerobic bacteria, thermophilic bacteria and thermodulic spores as shown in Table 1.
TABLE 1Number of viableSubject bacteriumbacteria cells per gAerobic bacteria9.9 × 108Thermophilic bacteria8.4 × 107Thermodulic spores2.8 × 107Enterobacteria100 or lessGram-negative bacteria100 or lessGram-positive bacteria2.8 × 106Lactic acid bacteria100 or lessAnaerobic bacteria100 or lessMesophilic actinomycetes1.1 × 103Thermophilic actinomycetes6.0 × 102Filamentous fungi100 or lessYeast100 or less
On the other hand, in the culture, a colony that grew dominantly on the culture plate was selected to obtain an isolated bacterium, and the isolated bacterium was subjected to morphological observation and the like in order to searched for microbes that may be concerned with fermentation. As a result, it was revealed that the following microbes are concerned with the fermentation.
TABLE 2Number of viableIsolated bacterium groupbacteria cells per gPolymorphic, sporeless gram-positive bacillus7 × 102Aerobic spore-forming bacteriamesophilic3 × 108thermophilic8 × 107Catalase-positive gram-positive cocci1 × 107Actinomycetesmesophilic1 × 103thermophilic6 × 102
As described above, it was revealed that mainly polymorphic, sporeless gram-positive bacilli, aerobic spore-forming bacteria (mesophilic and thermophilic) are involved.
On the other hand, measurement of thermophiles was performed referring to the description in, “Methods for Isolating Microbes”, YAMAZATO, Kazuhide and three others, ed., published by R&D Planning. The dominant thermophile was aerobic spore-forming bacteria (thermophilic).
Further, the mesophilic aerobic spore-forming bacterium (isolated bacterium a), thermophilic aerobic spore-forming bacterium (isolated bacterium b), and thermophile (isolated bacterium c) that were isolated predominantly in the above-mentioned search of microbes were subjected to morphological observation, physiological property tests and measurement of the GC content of DNA in bacterial cell. The results obtained are shown in Table 3.
TABLE 3Test resultIsolatedIsolatedIsolatedTest Itembacterium abacterium bbacterium cMorphologyrodrodrodGram stain+++Spore+++ShapeCircular toEllipsoidalEllipsoidalellipsoidalSiteCentralQuasi-Quasi-peritrichousperitrichous toperitrichousSporangiumNot swollenSwollenNot swollento slightlyswollenMotility−−+Behavior toward enzymesAerobicAerobicAerobicCatalase+++Growth under anaerobic−−−conditionsV-P reaction−−−pH of V-P broth6.58.0*25.6Acid formationGlucose−−*2−ArabinoseNT*−*2NT*XyloseNT*−*2NT*MannitolNT*−*2NT*Gas formation from glucose−−*2 −Casein decomposition+−NT*Gelatin salination+−+Starch decomposition−−−Assimilation of citrate−−*2−Assimilation of propionate−−*2−Tyrosine decomposition−−−Phenylalanine deamination−NT*NT*Egg yolk reaction−−−Nitrate reduction+−−Growth at pH 6.8+−+(Nutrient broth)Growth at pH 5.7−−−Growth in the presence of++−5% NaClGrowth in the presence of++−7% NaClGrowth at 10° C.−−NT*Growth at 30° C.+slow−Growth at 40° C.+++Growth at 50° C.−+NT*Growth at 55° C.NT*++Growth at 65° C.NT*−+Growth at 70° C.NT*NT*+Growth at 71° C.NT*NT*+Growth at 72° C.NT*NT*−GC content of cell DNA52*152*140*1(mol %)*NT: No test performed;*1By an HPLC method;*2Medium adjusted to pH 8.0 being used.
The isolated bacterium a did not correspond to any of the species in respect of the properties so that its species was not identified. The isolated bacterium b showed good growth on a slightly alkaline medium (pH 8.0 to 8.5) but did not grow on a medium at pH 7.0, and the results of tests on other properties suggested that it was a species close to Bacillus badius or B. brevis. However, bacterium b has properties which are non-typical to either of them, so that no identification of species was attained. Further, because the isolated bacterium c showed bacteriological properties identical to those of Geobacillus stearothermophilus, it may be identified as the same species. However, a great difference in their GC contents suggested that they are closely-related species.
These isolated bacteria have been deposited at Agency of Industrial Science and Technology, National Institute of Bioscience and Human-Technology (presently, International Patent Organism Depository, National Institute of Advanced Industrial Science and Technology), where accession numbers were respectively assigned: YM-01 accession number FERM P-15085 for the isolated bacterium a, YM-02 accession number FERM P-15086 for the isolated bacterium b, and YM-03 accession number FERM P-15087 for the isolated bacterium c.
The inventors of the present invention further made studies on the presence of such microbes that grow at high temperatures in compost and have surprisingly found a hyperthermophile belonging to a new genus that vigorously multiplies at high temperatures of 75° C. or more, still shows its multiplication at 85° C. but shows no multiplication at 50° C. or less.